Problem with Rosetta gami B protein expression!!!!

I am trying to express a disulfide rich antimicrobial peptide in the pRSET-A vector with His-tag in the Rosetta gami B DE3 pLysS bacterial host.

I saw that due to its trx/gor mutations it grows slowly than the BL21 cells as mentioned in the manual. So for the starter culture I added 40ul of a 20% glycerol stock with four antibiotics Tet,Kan,Cam (required for host) and Amp( for vector) in 10ml culture and incubated at 37C at 180rpm shaking overnight (18h) and seen good growth with an OD600=1.3.Then I inoculated 10ml LB with all the above mentioned antibiotics and 40ul of the starter culture and rotated at 180rpm.I started the OD monitoring and saw no growth at all after 2 hours and then increased the speed to 200rpm and after 4 hours still no growth.I needed an OD600=0.5-0.6 for IPTG induction.I let it shake at 200rpm for almost 24 hours and still saw little growth. I also tried it with Terrific Broth but it showed worse than LB, even though its a richer medium.I dont think cells didnt grow due to my protein being an antimicrobial peptide, as the starter culture grew nicely and I didnt even start induction.What is the problem with my approach?

People who have experience with my host bacteria please help me as how I can reach OD600=0.5-0.6 and in what time and conditions.I am completely new to this and this is first time its done in my lab.

Origami cells are notoriously challenging to work with. For starters, you shouldn't need Tet and Kan in the cultures since these are selection markers encoded in the genome. They are good to use when initially plating your cells for strain confirmation but are not necessary for strain propagation. The tetracycline will certainly decrease your growth rate more than any other antibiotic. So I suggest only having Cam for maintaining the pLysRare plasmid and Amp for maintaining your plasmid of interest when growing your expression culture.

Other options to think about:

1. 40ul into 10ml is a very small inoculum even for well-behaved strains. Given your slow rate of growth, try 0.5 - 1 ml (5-10% inoculum).

2. Why not add glycerol to your expression culture? It seems to help your starter culture, and glycerol will not interfere with target protein expression (in fact it is often added to a lot of expression media as an extra carbon source).

4. Try auto-induction media. This way you can just inoculate and come back in 24-48 hrs. No need to stand around impatiently waiting for cells to reach a certain OD to express...the cells will express on their own when they get to the right OD.

5. Try a traditional cell line and express your protein in the periplasm for disulfide formation. Yields are low with periplasm expression but a whole lot better than nothing in a cell line that doesn't grow.

6. If you're growing your BL21 cells with the pRSET vector from a pre-made frozen glycerol stock, try starting from a fresh transformation.

7. Simplify your expression system and still meet your needs...for example, (i) move your gene to a pBAD vector which is less leaky than T7 systems so you don't need the pLysS functionality, and (ii) codon optimize your gene and order the Gblock from IDT for cheap so that you don't need the pRare system....and then express in Origami2(DE3) or OrigamiB(DE3). This reduces the complexity of your expression system and your cells only need to maintain 1 plasmid.

8. There is always the option of expressing in a non-Origami strain and refolding from inclusion bodies (assuming your protein goes into inclusion bodies if the proper disulfides are not made). Refolding from inclusion bodies can be a pain especially if you need proper redox buffering for disulfide shuffling/formation....however you can get pretty good yields.